Portable surface plasmon resonance biosensor

ABSTRACT

A surface plasmon resonance biosensor device and system are provided. The simplicity of SPR biosensor design allows easy integration with both QCM and electrochemistry techniques, not found in current SPR biosensor devices. In some embodiments, the surface plasmon resonance biosensor device has a dual SPR/QCM sample holder which allows simultaneous detection by both surface plasmon resonance and also quartz crystal microbalance (QCM) techniques. In additional embodiments, the surface plasmon resonance biosensor device and/or the dual SPR/QCM technique can be integrated with electrochemistry techniques by incorporate reference and counter electrodes in the SPR or SPR/QCM sample holder. Methods of using the device and system to determine whether an analyte of interest is present in a sample are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

Not Applicable

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not Applicable

BACKGROUND OF THE INVENTION

(1) Field of the Invention

The present invention relates generally to surface plasmon resonance.Specifically, the present invention relates to a simple and reliablesurface plasmon resonance biosensor device.

(2) Description of the Related Art

Life phenomena are results of biomolecular interactions. Biomolecularinteractions are traditionally studied using techniques such asenzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), andaffinity chromatography. Surface plasmon resonance (SPR) biosensingtechnique provides two main advantages over these techniques. First, thebiomolecular interactions can be monitored in real-time. Second, it isnot necessary to label the interacting biomolecules. Surface plasmon isa quantum name for an electric charge density wave that propagates on aninterface between a metal and a dielectric, just like photon is aquantum name for a light wave. Surface plasmons are described in U.S.Pat. No. 7,084,980 to Jones, et al. hereby incorporated herein byreference in its entirety. Surface plasmons resonate upon excitation byelectromagnetic radiation entering an interface of metallic material anda dielectric material. The surface plasmon responds to changes in theenvironment in close proximity to the interface. This fact makes surfaceplasmon resonance useful for the detection of biomolecular interactions.A practical and commonly used method by which to excite the surfaceplasmon was initially suggested by Kretschmann (Kretschmann, E. (1971)“Die Bestimmung optischer Konstanten von Metallen durch Anregung vonOberflachenplasma-schwingungen.” Z. Phys., 241: 313-324). In theKretschmann configuration, a prism is used as a coupler between incidentphotons and surface plasmons on a surface of a thin metal filmevaporated onto the prism. Since the refractive index of a prism isusually higher than the refractive index of its ambient environment (airor water), there is a critic incident angle θ_(c) for the lightreflected inside the prism. Under the conditions when θ≧θ_(c), totallight reflection occurs inside the prism. For p-polarized light, theincident photons can excite surface plasmons on surface of a metal film.The incident angle for surface plasmon resonance is called resonanceangle (θ_(SPR)). Since there is energy transfer from photons to surfaceplasmons during resonance, the reflectivity of incident light couldchange from 1 to 0 at θ_(SPR). A graph of the relationship betweenreflectivity and incident angle is called an SPR spectrum.

Under some conditions, the shift of the resonance angle Δθ_(SPR) isproportional to (n_(s)−n_(a))l, where n_(s) and l are sample refractiveindex and thickness, n_(a) is the ambient refractive index. From thelinear relationship, the amount of protein adsorbed on a gold film canbe detected at as low as 0.01 ng/mm², and the affinity ofligand-receptor interaction can be detected. After Nylander (Nylander,C., Liedberg, B. & Lind, T. (1982/83), “Gas detection by means ofsurface plasmon resonance” Sensors and Actuators, 3: 79-88) publishedthe first paper about SPR biosensing in 1983, a project was initiated atPharmacia of Sweden (Liedberg, B., C. Nylander, et al. (1995).“Biosensing with surface plasmon resonance—how it all started”,Biosensors and Bioelectronics 10(8): i-ix.) in 1984. In 1986 a separatecompany, Pharmacia Biosensor, was formed for the development of the newbiosensor technology. Today the successful Pharmacia Biosensor Companyis called Biacore (http://www.biacore.com) and it makes many kinds ofcommercial SPR biosensors. For biologists, these Biacore SPR biosensorsare very useful, but also very expensive. The price of the latest modelBiacore 3000 is presently about three hundred thousand dollars and theprice of a disposable sensing chip is about one hundred and twentydollars. A fully used Biacore SPR biosensor can consume thousands of thesensing chips per year.

U.S. Pat. Nos. 6,493,097 and 6,714,303 to Ivarsson et al. teach anapparatus and method of examining thin layer structures on a sensorsurface by imaging light reflected by the surface during SPR microscopy.The apparatus uses a light source which illuminate collimator optics toproduce a parallel light beam. The light beam passes an interferencefilter as a monochromatic beam and impinges on two flat scanner mirrorsbefore the light beam is deflected into a prism or grating to the sensorsurface. An optical system then produces an image of the sensor surfaceat a detector. The optical system adds cost and complexity to theapparatus.

While the related art teach surface plasmon resonance biosensors, therestill exists a need for an inexpensive and portable surface plasmonresonance biosensor.

OBJECTS

Therefore, it is an object of the present invention to provide aninexpensive and portable surface plasmon resonance biosensor device.These and other objects will become increasingly apparent by referenceto the following description.

SUMMARY OF THE INVENTION

The present invention provides a surface plasmon resonance biosensordevice for analysis of a fluid sample comprising: a support means; acapacitive angle sensor mounted to the support means having a shaft; arotatable adapter means rotatably mounted on the shaft; a prism or ahalf cylinder mounted on the rotatable adapter means; a light sourcemounted to the support means, positioned to project a beam of lightthrough a first side of the prism or half cylinder to a second side ofthe prism or half cylinder; a thin metallic film with an inner surfaceaffixed on or adjacent to the prism or half cylinder; a sample holderhaving a sealing means removably sealed against an exposed surface ofthe thin metallic film, the sample holder, sealing means and thinmetallic film defining a sample chamber; a light response element as atransducer to generate an intensity output mounted on a third side ofthe prism or half cylinder, wherein when the sample is provided to thesample chamber and the light source projects the beam of light, thecapacitive angle sensor measures an incident angle of the beam of lightupon the metallic film to provide an incident angle output and the lightresponse element measures light intensity reflected from the innersurface of the thin metallic film to provide the intensity output.

In further embodiments, the metallic film is gold. In still furtherembodiments, the prism or half cylinder is made of glass or plastic. Instill further embodiments, the light response element is a solar cell.In still further embodiments, the light source is a laser apparatus. Instill further embodiments, the rotatable adapter means is a rotatableplate. In still further embodiments, the sealing means is an o-ring. Instill further embodiments, the support means is a mounting table. Instill further embodiments, the surface plasmon resonance biosensordevice further comprises a low speed motor rotatably engaged with therotatable adapter means to turn the rotatable adapter means duringanalysis of the fluid sample. In still further embodiments, the metallicfilm is provided upon a transparent cover. In still further embodiments,the sample holder is a dual SPR/QCM sample holder which allowssimultaneous detection by both surface plasmon resonance and also quartzcrystal microbalance (QCM) techniques.

The present invention provides a surface plasmon resonance biosensorsystem for analysis of a fluid sample comprising: a support means; acapacitive angle sensor mounted to the support means having a shaft; arotatable adapter means rotatably mounted on the shaft; a prism or ahalf cylinder mounted on the rotatable adapter means; a light sourcemounted to the support means, positioned to project a beam of lightthrough a first side of the prism or half cylinder to a second side ofthe prism or half cylinder; a thin metallic film with an inner surfaceaffixed on or adjacent to the prism or half cylinder; a sample holderhaving a sealing means removably sealed against an exposed surface ofthe thin metallic film, the sample holder, sealing means and thinmetallic film defining a sample chamber; a light response element as atransducer to generate an intensity output mounted on a third side ofthe prism or half cylinder, wherein when the sample is provided to thesample chamber and the light source projects the beam of light, thecapacitive angle sensor measures an incident angle of the beam of lightupon the metallic film to provide an incident angle output and the lightresponse element measures light intensity reflected from the innersurface of the thin metallic film to provide the intensity output; ananalog to digital (A/D) data acquisition device electrically connectedto the capacitive angle sensor and the light response element forgenerating a data signal when the sample is provided to the samplechamber from an incident angle output of the capacitive angle sensor andan intensity output from the light response element; and a personalcomputer electrically connected to the data acquisition device toreceive and process the data signal generated by the data acquisitiondevice and provide a surface plasmon resonance spectrum when the fluidsample is provided to the sample chamber.

In further embodiments, the metallic film is gold. The surface plasmonresonance biosensor device of Claim 12, wherein the metallic film isgold. In still further embodiments, the prism or half cylinder is madeof glass or plastic. In still further embodiments, the light responseelement is a solar cell. In still further embodiments, the light sourceis a laser apparatus. In still further embodiments, the rotatableadapter means is a rotatable plate. In still further embodiments, thesealing means is an o-ring. In still further embodiments, the supportmeans is a mounting table. In still further embodiments, the surfaceplasmon resonance biosensor further comprises a low speed motorrotatably engaged with the rotatable plate to turn the plate duringanalysis of the sample. In still further embodiments, the sample holderis a dual SPR/QCM sample holder which allows simultaneous detection byboth surface plasmon resonance and also quartz crystal microbalance(QCM) techniques.

The present invention provides a method to determine whether an analyteof interest is present in a fluid sample comprising: providing a surfaceplasmon resonance biosensor device comprising a support means; acapacitive angle sensor mounted to the support means having a shaft; arotatable adapter means rotatably mounted on the shaft; a prism or ahalf cylinder mounted on the rotatable adapter means; a light sourcemounted to the support means, positioned to project a beam of lightthrough a first side of the prism or half cylinder to a second side ofthe prism or half cylinder; a thin metallic film with an inner surfaceaffixed on or adjacent to the prism or half cylinder; a sample holderhaving a sealing means removably sealed against an exposed surface ofthe thin metallic film, the sample holder, sealing means and thinmetallic film defining a sample chamber; a light response element as atransducer to generate an intensity output mounted on a third side ofthe prism or half cylinder, wherein when the sample is provided to thesample chamber and the light source projects the beam of light, thecapacitive angle sensor measures an incident angle of the beam of lightupon the metallic film to provide an incident angle output and the lightresponse element measures light intensity reflected from the innersurface of the thin metallic film to provide the intensity output;providing the fluid sample to the sample chamber; turning the rotatableadapter means; detecting the reflected light intensity with the lightresponse element to provide the intensity output; detecting the incidentangle with the capacitive angle sensor to provide the incident angleoutput; processing the intensity output and the incident angle output toprovide an surface plasmon resonance spectrum; analyzing the peak of thesurface plasmon resonance spectrum to determine whether the analyte ispresent in the sample.

In further embodiments, the sample holder is a dual SPR/QCM sampleholder, further comprising the steps of analyzing signals from areference electrode and a counter electrode on the dual SPR/QCM sampleholder for electric deposition of polymers and sample reduce/oxidation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of one embodiment of a preferred portablesurface plasmon resonance biosensor device 10 of the present invention.

FIG. 2 is a top view of the surface plasmon resonance biosensor device10 of FIG. 1.

FIG. 3 is a cross-sectional view of the surface plasmon resonancebiosensor device 10 of FIG. 2 taken along line 3-3.

FIG. 4 is a schematic representation of a top view the surface plasmonresonance biosensor device 10 of FIG. 1 illustrating the incident angleθ of the laser beam 42 passing through the first side 31 to the secondside 32 of the prism 30 and reflecting through the third side 33 ontothe solar cell 80.

FIG. 5 is a schematic representation of a second embodiment of a surfaceplasmon resonance biosensor device 10′ that electronically displaysincident angle θ and R. A single chip computer is included inside theembodiment, so it can work without a personal computer.

FIG. 6 is a top view of a third embodiment of a surface plasmonresonance biosensor device 110 of the present invention having alow-speed motor to turn the rotatable plate 120 in small increments.

FIG. 7 is a top view of a fourth embodiment of a surface plasmonresonance biosensor device 210 of the present invention set up as an SPRmicroscope.

FIG. 8 is another embodiment of a surface plasmon resonance biosensorsystem 300 having the surface plasmon resonance biosensor device 10 ofFIG. 1 electrically connected to a personal computer 320 to receive andprocess data from a data acquisition device 310.

FIG. 9 illustrates a magnified view of the optical media and opticalpath of the plasmon resonance biosensor device 10 of FIG. 1.

FIG. 10 is a schematic block diagram 400 of the software setup whichprovides the surface plasmon resonance spectra from the surface plasmonresonance biosensor device 10.

FIG. 11 is a display screen of the interface 510 generated by thesoftware illustrated schematically in FIG. 10.

FIG. 12A is a graph illustrating a total reflection curve and a SPRspectrum in air.

FIG. 12B is a graph of three SPR spectra (reflected light intensityversus the outside incident angle φ) directly measured by the surfaceplasmon resonance biosensor 10 of FIG. 1.

FIG. 12C is a graph of three surface plasmon resonance spectra(reflected light intensity versus the inside incident angle θ) that isderived from FIG. 12B.

FIG. 13 is a graph of three surface plasmon resonance spectra measuredby the SPR biosensor device 10 of biological samples. IgG antibodyspecifically binds to Protein A immobilized on the gold surface of themetallic film immersed in Phosphate Buffered Saline (PBS) buffer in thesample chamber 78. “PBS Buffer”: PBS sample buffer alone. “Protein A”:Protein A immobilized on the gold surface in a PBS sample buffer. “IgG”:IgG antibody binding to Protein An immobilized on the gold surface in aPBS sample buffer.

FIG. 14 is a standard plot using experiment data from the surfaceplasmon resonance biosensor 10 of FIG. 1. This linear relationship shownin the graph can be used to measure refractive index of samples

FIG. 15 is the molecular formula of a synthesized lipid mannose-SH usedas a capture reagent.

FIG. 16 is a three-dimensional model of the synthesized lipid mannose-SHmolecule.

FIG. 17A is a graph illustrating mannose-SH lipid binding to a goldsurface of the surface plasmon resonance biosensor 10 of FIG. 1 whilethe liquid in the chamber is ethanol. During measurements, the incidentangle φ was fixed at 26.5°. FIG. 17B is a graph showed the SPR spectrashift caused by Mannose-SH lipid in ethylnol.

FIG. 18A and FIG. 18B are graphs illustrating protein Concanavalin A(Con A) in PBS buffer binding to the mannose-SH immobilized on the goldsurface of the surface plasmon resonance biosensor 10 of FIG. 1. Duringmeasurements for FIG. 18A, the incident angle φ was fixed at 29.0°.

FIG. 19A and FIG. 19B are graphs illustrating E. coli K12 binding to theCon A surface (immobilized on the mannose-SH lipid monolyer on the goldsurface of the surface plasmon resonance biosensor 10 of FIG. 1) ifthere are ConA molecules in the liquid. During measurements for FIG.19A, the incident angle φ was fixed at 28.0°.

FIG. 20 is a top view of the V-shaped holder 25 of the surface plasmonresonance biosensor device 10 with a dual SPR/QCM sample holder 610replacing sample holder 70.

FIG. 21 is a front view of the dual SPR/QCM sample holder 610 of FIG.20.

FIG. 22 is a cross-sectional view of the dual SPR/QCM sample holder 610taken along line 22-22 of FIG. 21.

DETAILED DESCRIPTION OF THE INVENTION

All patents, patent applications, government publications, governmentregulations, and literature references cited in this specification arehereby incorporated herein by reference in their entirety. In case ofconflict, the present description, including definitions, will control.

The term “beam of light” as used herein refers to light such as, but notlimited to, a beam of laser light.

The term “capture reagent” as used herein refers to any molecule boundto the metallic film that is used to bind an analyte of interest.

The term “prism” or “half cylinder” as used herein refers to any opticalmaterial (glass or plastic) having a refractive index above 1.5 withmulti-side or a half cylinder geometric shapes. The present examples aredirected to a triangular prism, however the term “prism” is not limitedto a triangular prism.

The term “light response element” as used herein refers to any lighttransduction apparatus that can detect light and generate an output inresponse to the light intensity. Any means for transducing light knownin the art, such as, but not limited to a solar cell is encompassed bythe term.

The term “light source” as used herein refers to an apparatus forgenerating monochromatic light such as, but not limited to, a laserapparatus.

The term “rotatable adapter means” as used herein refers to anyapparatus for mounting the prism to the capacitive angle sensor of thesurface plasmon resonance device. The term “rotatable adapter means” caninclude an means for adapting such as, but not limited to, a rotatableplate.

The term “sealing means” as used herein refers to any means for sealingknown in the art, such as, but not limited to an o-ring or gasket.

The term “SPR” as used herein refers to surface plasmon resonance.

The term “support means” as used herein refers to any apparatus forsupporting components of the surface plasmon resonance device such as,but not limited to, a mounting table, surface, stand or other holderknown in the art.

The improved portable and economic SPR biosensor of the presentinvention is a significant improvement of the SPR biosensor thatinventor Dr. Xiao Caide described in his Ph.D. thesis work (Caide, X.and F. Sui Sen (1999), “Numerical simulations of surface plasmonresonance system for monitoring DNA hybridization and detectingprotein-lipid film interactions,” European Biophysics Journal 28(2):151-157.) in China between 1990 and 1996. The improved SPR biosensor ofthe present invention can be used both for research and educationalpurposes.

One embodiment of the surface plasmon resonance biosensor device 10 ofthe present invention is illustrated in FIGS. 1-3. In this embodiment ofthe device 10, a mounting table 12 with a top surface 13 and a bottomsurface 14 (FIG. 3) is used to support the components. A shaft 16, asseen in FIG. 3, of a capacitive angle sensor 90 is mounted through ahole (not shown) in the mounting table 12. One commercially availablecapacitive angle sensor that can be used is the Series 600 AngularDisplacement Transducer (ADT) Model No. 0601-0000 (Transtek Inc.,Ellington, Conn.). The capacitive angle sensor 90 is capable ofmeasuring the rotation of the shaft 16 relative to the mounting table 12to provide a first signal output. A rotatable plate 20 is mounted overthe table 12 affixed on the top end 17 of the shaft 16. The bottom end(not shown) of the shaft 16 extends down into the body of the capacitiveangle sensor 90 mounted on the bottom surface 14 of the mounting table12. The rotatable plate 20 is free to turn with respect to the mountingtable 12 as illustrated by the arrow in FIG. 2. On the top of therotatable plate 20 is a depressed portion 20A disposed between twoprojecting ridges 20B, 20C at opposed ends of the rotatable plate 20. Anadjustable base 21 having two slots 22 at opposed ends of therectangular shaped adjustable base 21 is adjustably attached by a firstset of Allen screws 23 in the depressed portion 20A of the rotatableplate 20 between the two projecting portions 20B, 20C. A V-shapedtransparent holder 25 is mounted on the adjustable base 21 by means of asecond set of Allen screws 26. A prism 30, for example a ZF7 glassuncoated right-angle prism (Red Optronics, Mountain View, Calif.), isplaced within the V-shaped transparent holder 25 on the rotatable plate20. A laser apparatus 40, for example a Melles Griot (Carlsbad, Calif.)06 DAL 103 laser, is affixed to the top surface 13 of the table 12,positioned to project a beam of laser light 42 (FIG. 3) through a firstside 31 of the prism 30 to a second side 32 as the base of the prism 30.The adjustable base 21 can be moved in a direction along the rotatableplate 20 to move the second side 32 of the prism 30 forwards orbackwards so as to center it on the rotatable plate 20. Once the prism30 is at the desired location, the first set of Allen screws 23 can thenbe tightened to secure the adjustable base 21 to the rotatable plate 20.

FIG. 3 illustrates the device set up to receive a fluid sample. Atransparent cover 50 (illustrated in FIG. 3) is removably mounted to theprism 30 with a first side 51 of the transparent cover 50 mountedagainst the second side 32 of the prism 30. A thin coating of immersionoil 53 (illustrated with exaggerated thickness in FIG. 3) can be used tomake a close seal between the prism 30 and the transparent cover 50. Thetransparent cover 50 has a thin metallic film 60 having an inner surface61 affixed to a second side 52 of the transparent cover 50. Thetransparent cover 50 with the affixed thin metallic film 60 can bedisposable. Thus, the surface plasmon resonance biosensor device 10 canbe reused with a new and clean thin metallic film 60 after each use. AnSPR sample holder 70 having an o-ring 76 is removably sealed against anexposed surface 62 of the thin metallic film 60. The SPR sample holder70 holds the o-ring 76 firmly against the thin metallic film 60 by ascrew 28 threaded through a hole 27A in a back plate 27. A user canthereby add pressure to secure the sample holder 70 by gripping the knob29 and twisting the screw 28. When the screw 28 is tightened, the SPRsample holder 70, o-ring 76 and thin metallic film 60 form a samplechamber 78. A fluid sample can be placed into the sample chamber 78through an opening created by a wedge-shaped cut 72 in the SPR sampleholder 70. A solar cell 80, such as a Radio Shack® (Fort Worth, Tex.)silicon solar cell model 276-124, is mounted on the V-shaped transparentholder 25. The solar cell 80 is mounted adjacent to a third side of theprism 30 for measuring the laser light 42 intensity reflected from theinner surface 61 of the thin metallic film 60 and through the third side33 of the prism 30.

FIG. 4 is a schematic representation of a top view the surface plasmonresonance biosensor device 10 of FIGS. 1-3. The laser apparatus 40 issecured to the top surface 13 of the mounting table 12. When the laserapparatus 40 is activated, a beam of laser light 42 is emitted by thelaser apparatus 40 which penetrates into the first side 31 of the prism30. The beam of laser light 42 is then reflected back from the multiplelayers shown in FIG. 9, penetrates the third side 33 of the prism andreaches the solar cell 80. The solar cell 80 transduces the reflectedlaser light 42 intensity into an electrical signal to provide anintensity output. FIG. 4 illustrates the incident angle θ, relative to aline 34 normal to the second side 32 of the prism, made by the laserbeam 42 as it passes through the first side 31 to the second side 32 ofthe prism and reflects back through the third side 33 onto the solarcell 80. The capacitive angle sensor 90 (see FIGS. 1 and 3) mounted onthe second end of the shaft 16 measures the incident angle θ (FIG. 4) ofthe laser light 42 with the thin metallic film 60 to provide an incidentangle output.

To measure SPR spectra in a SPR biosensor device 10 with a monochromaticlight source, a user must change the incident angle θ of the beam oflaser light 42 in FIGS. 1-3. In the Biacore® system, a focused lightbeam is used, so that the incident light with different incident anglecan be detected by a one dimensional light sensitive diode array. Thisoptical system is expensive. In the SPR biosensor Dr. Xiao made inTsinghua University (China), a θ-2θ goniometer from an x-ray diffractionmachine was used to trace the reflected light spot, because the anglebetween the incident light and the reflected light changes 2dθ if theincident angle changes de. Goniometers used with x-ray diffractionmachines are instruments used for precise measurement of angles ofcrystals. However, these goniometers have the drawbacks of being veryexpensive, heavy (>100 kg) and they give no electric signal thatprovides angle shift information.

In the portable SPR biosensor device 10 of the present invention, acommercial capacitance angle sensor 90 is used to measure the incidentangle θ. With a fifteen volt direct current (15V DC) input, thecapacitance angle sensor 90 can give an angle signal output of onehundred millivolts per degree (100 mV/Degree). The capacitance anglesensor 90 and a semiconductor laser 40 were both affixed to the body ofan aluminum mounting table 12 as illustrated in FIGS. 1-3. The prism 30,made of N—SF6 (ZF7) glass, was fixed on the horizontal disk-shapedrotatable plate 20 coupled to the shaft 16 of the capacitance anglesensor 90. The rotation of the shaft 16 of the capacitance angle sensor90 changes the incident angle θ of the beam of laser light 42 reflectedinside of the prism 30. The reflected laser light 42 illuminates a spoton the solar cell 80 that will also move a distance of r×Δθ, where r isthe distance between the solar cell 80 and the reflective laser light 42spot in the prism 30, and Δθ is the angle shift for an SPR spectrum.Usually Δθ is less than 18°, r less than 30 mm, and the distance of thelight spot on the moving solar cell 80 will be less than 10 mm. Thereflected laser light 42 spot will not move off of the solar cell 80that is turning with the prism 30. Tracing the reflected light spot inSPR biosensors is a novel part of the design. No expensive optical ormechanical devices are needed in the SPR biosensor device 10. FIG. 5 isa schematic representation of a second embodiment of a surface plasmonresonance biosensor device 10′, otherwise identical to the surfaceplasmon resonance biosensor device 10 illustrated in FIGS. 1-3,additionally having displays for incident angle θ and R.

FIG. 8 is one embodiment of a surface plasmon resonance biosensor system300 having the surface plasmon resonance biosensor device 10 of FIG. 1electrically connected to a personal computer 320 to receive and processdata from a data acquisition device 310. There are two analog DC signaloutputs from the SPR biosensor device 10. The first signal output isfrom the capacitance angle sensor 90, and the second signal output isfrom the solar cell 80. It is easy to use any commercially available USBdata acquisition device 310, such as a Personal Measurement Device™brand USB-based analog and digital I/O module PMD-1608FS (MeasurementComputing Corporation, Norton, Mass.) to record the two analog signalsfor collecting an SPR spectrum. LabVIEW® development software (NationalInstruments, Austin, Tex.) can be used to write software to control theSPR biosensor device 10, however other software can be used. The opticallayout of the SPR biosensor device is illustrated in the magnified viewof FIG. 9. A transparent cover 50 of glass, gold (Au) as the thinmetallic film 60, and a bound sample layer 66 constituted a sensitivechip. The chip was stuck to the base, that is the second side 32, of theprism 30 by immersion oil 53 (see FIG. 3). A rubber o-ring 76 andTeflon® polytetrafluoroethylene (PTFE) chuck as the sample holder 70were pressed to the sensitive chip, thereby forming a two hundredmicroliter (200 μl) volume sample chamber 78 (see FIG. 3). In someembodiments, there can be two channels in the sample holder 70 forpumping a fluid sample (ie. liquid or gas) into the sample chamber 78.In the SPR system shown in FIG. 9 there are five optical medium: theprism 30, the transparent cover 50, the gold as the thin metallic film60, the bound sample layer 66 and the liquid buffer 68. The prism 30 wasmade from a piece of ZF7 glass. Its refractive index value at 650 nm was1.798. The refractive index of microscope glass as the transparent cover50 was 1.56. The refractive index of the gold as the metallic film 60 at650 nm was 0.204+i3.484. The buffer refractive index was chosen as 1.331for water at room temperature.

While measuring the spectra shown in FIG. 12C, there was no sample layer66 film on the gold metallic film 60 surface of the sensing chip. Air,water or alcohol as the media was in direct contacted with the chip. Bymanually turning the rotatable plate 20 illustrated in FIG. 1-FIG. 3,the SPR spectra was obtained. The resonance angle (θ_(SPR)) increasesfrom 36.34°, 53.49° to 55.26° in response to the refractive index changeof the media contacting the gold metallic film 60 surface from 1.000,1.331 to 1.360. From our calculations, the SPR biosensor device 10 canbe used to measure refractive index (n, 1.0 to 1.4) of both gases andliquids as the fluid sample using the standard plot of FIG. 14. Therelationship between θ_(SPR) and n is shown in Equation 1.

n=1.549 sin(θ_(SRP))+0.095  Equation 1

FIG. 12A is a graph of a total reflection curve and a SPR spectrum inair measured by the SPR biosensor device 10 of the present inventionillustrated in FIG. 1-FIG. 3. FIG. 12B is a directly measured graph ofreflected light intensity versus the outside incident angle φ, whileFIG. 12C is a graph of the surface plasmon resonance spectra. FIG. 12Cshows three SPR spectra measured by the SPR biosensor device 10 of thepresent invention for air, water and alcohol. FIG. 13 shows SPR spectraof biological molecular interactions on a gold metallic film 60 surfaceimmersed in phosphate buffered saline (PBS) buffer. After the SPRspectrum of the gold metallic film 60 in the PBS buffer was obtained(labelled “PBS Buffer”), Protein A solution (10 μL×2 mg/mL) was injectedinto the sample chamber 78 with 150 μL of PBS buffer. Ten minutes later,the sample chamber 78 was washed and refilled with PBS buffer, and theSPR spectrum labelled “Protein A” was measured. At last 10 μl, goatserum with antibody was injected into the sample chamber 78. Afterwashing and refilling the sample chamber 78 with PBS buffer, the SPRspectrum labelled “IgG” was obtained. We can see that protein moleculesadsorbed on the gold surface shifted the SPR spectra from left to right.From numerical simulations it was found that there was a linearrelationship between protein surface concentration Γ (ng/mm²) and SPRresonance angle shift θ_(SPR) (degree).

Γ=8.55Δθ_(SPR) or Γ=−4.70Δφ_(SPR)

With these formula, the portable SPR biosensor can measure affinityconstants about ligand-receptor interactions. In one embodiment, theincident angle θ is changed manually. The SPR biosensor device 10 inthis embodiment can be used to use as a teaching instrument to teachstudents about SPR and how to measure liquid refractive index andantigen-antibody interactions. Optionally, in a second embodiment asillustrated in FIG. 6, a low speed motor 116 can be installed as a drivesystem 115 on the aluminum table 12 shown in FIG. 1-FIG. 3, so the scanangle can be automatically processed. The low speed motor 116 is mountedin a depression 114 in the table 12 by means of a mount 117. A gear box118 transmits power to a screw gear 119 that meshes with teeth 121 onthe outer perimeter of the rotatable plate 120. Otherwise the biosensordevice 110 is identical to the biosensor device 10 of FIG. 1-FIG. 3.With automatic angle scanning and any commercially available liquidinjection system (not shown), the homemade SPR biosensor device 110 canbe used as a professional research instrument in universities andhospitals. For example, the SPR biosensor device 10 can be used todetect tumor marker proteins or AIDS virus proteins in a patient's bloodserum within one minute without the necessity of labeling or treatingthe sample. Only ten microliters (10 μL) of serum is enough to use inmeasurements for clinical diagnosis.

Software for analysis of output signals was written with Labview® forPC. The interface 510 shown in FIG. 11 was obtained by pressing the“Print Scrn” key on the PC computer 320 keyboard after measurement withgold as the metallic film 60 with air in the sample chamber 78. On theupper-left corner of the interface 510 there is a light sensor graph 512which records the relationship between the solar cell voltage and time.The time interval 514 of each point on the light sensor graph 512 is 0.2seconds as shown on the lower-left corner of the interface. However, theinterval can be changed by the user. Below the light sensor graph 512there is a virtual meter called the outside angle meter 516. The outsideangle is the angle φ between the laser and the base of the prism 30 asillustrated in FIG. 9. The outside angle φ is the angle directlymeasured by the angle sensor 90 shown in FIG. 1. The relationshipbetween φ and the inside angle θ is:

$\begin{matrix}{\theta = {{45{^\circ}} + {{arc}\; {\sin \left\lbrack {\frac{1}{n_{0}}{\sin \left( {{45{^\circ}} - \phi} \right)}} \right\rbrack}}}} & {{Equation}\mspace{14mu} 3}\end{matrix}$

where the parameter n_(o) is the refractive index of the prism. At λ=650nm, the refractive index of the prism is 1.798. The measurement isstarted by pressing the arrow tool button 517 on the second row of thesoftware interface, as shown in FIG. 11. The angle φ was changed by theleft hand of the user, and both the angle and the reflected lightintensity is seen in real time. After the stop button 518 on thelower-middle of the interface is pressed, the measurement is stopped.The angle φ and light intensity parameters are combined to form the SPRspectrum shown in the XY graph 520 on the right of the screen. Fromoptical theory the total reflection angle of the system should beθ_(c)=arcsin(1/n_(o))=33.792°. From FIG. 11, the outside totalreflection angle φ_(c)=64.000°. According to Equation 3, the measuredtotal reflection angle is θ_(c)=33.012°. The difference between thetheory and measurement is 0.780°. The error might come from the anglesensor position for φ=0°, because it is difficult to exactly find 0° byeye.

The SPR vi Block Diagram 400 is illustrated in FIG. 10. To provide atime output 410, a white-loop 412 is performed by setting an intervaltime 413 and a time delay 414 which will stop cycling when the stopbutton 411 on the front interface is pressed. A white-loop cyclesequence number (i) 415 is set. The output time (t) 410 is then computedby the formula: t=i×interval time. The interval of each cycle is 0.2seconds by default, however users can change the interval. In eachcycle, the program reads the reflected light intensity from the solarcell 80 and the prism 30 position from the capacitance angle sensor 90.

The light intensity output 420 is provided by setting the hardwarenumber (“BoardNum”) 421 from the USB-1608FS data acquisition device(DAQ), channel number (“Channel”) 422, and analogous signal range (−1Vto +1V) 423. Next, a sub-program 424 from the USB-1608FS dataacquisition device which has eight channels (0, 1 . . . 7) for analogsignal input. The sub-program 424 converts the analog voltage on channel0 (the solar cell 80) to a 16-bit digital number. Then anothersub-program 425 converts the 16-bit digital number to a scientificnumber for voltage. The next light sensor sub-program 426 displays anintensity versus time (R-t) graph on the front interface.

The angle output 430 is provided by setting the hardware number(“BoardNum”) 431 from the USB-1608FS data acquisition device (DAQ),Channel Number (“Channel”) 432 for the angle sensor, and analog signalrange (“Range”) 433 from −10V to +10V. A sub-program 434 from USB-1608FSdata acquisition device converts the analog voltage on channel 1 (ie.the angle sensor 90) to a 16-bit digital number. Next, a sub-program 435converts the 16-bit digital number to a scientific number in voltage. Anamplification setting 436 of 10 to the sub-program 437 will result inthe angle sensor 90 providing a one volt direct current (1.0V DC) signalif the angle of the shaft rotates ten (10) degrees. The sub-program 437converts voltage to degrees. The outside angle sub-program 438 thendisplays angle versus time (angle-t) graph on the front interface.During an experiment, data provided at the light intensity output 420,the prism angle output 430, and the time data output 410 are each storedin a memory buffer. After the stop button 411 on the front interface ispressed, the light intensity data and prism angle data are combined todraw an SPR spectrum (“XY Graph”) 440. The light intensity, prism angleand time data are combined to a data sheet 450 for file recording.

The portable SPR biosensor device (10, 110) with modifications, can alsobe used as a surface plasmon microscope (SPM) (Rothenhäusler, B. and W.Knoll, Surface plasmon microscopy. Nature, 1988. 332: p. 615-617). FIG.7 is a top view of a fourth embodiment of a surface plasmon resonancebiosensor device 210 of the present invention set up for use as an SPRmicroscope. By using a lens 280 (focus ˜20 mm) instead of the solar cell80, and using a CCD camera 281 (2000 mm away), a ×100 microscope basedon surface plasmon resonance is provided. (Zhou, Y., X. Caide, and S. F.Sui, Assembly of supported membranes studied by surface plasmonmicroscopy. Molecular Crystals And Liquid Crystals Science AndTechnology Section A Molecular Crystals And Liquid Crystals, 1999. 337:p. 61-64). The lens 280 projects laser light to the CCD camera 281adjustably mounted on rollers 282 in rails 284, so that the CCD camera281 can be moved nearer or farther from the lens 280. The rails 284 aremounted on elevator blocks 285 to the rotatable plate 20 describedpreviously. When the CCD camera 281 is at the desired distance from thelens 280, it can be locked in place with a locking knob 283. This fourthembodiment of the SPM biosensor device 10 can be used to directly see amolecular monolayer in real time.

The portable SPR biosensor device 10, 110 of the present invention canbe easily integrated with various electrochemical techniques, such asvoltammetry. In this hyphenated technique, the SPR gold electrode can bealso used as working electrode for an electrochemistry study. As aresult, additional control and information can be applied and obtainedfrom the gold SPR electrode. The combination of SPR withelectrochemistry can provide correlations between electrochemicallyinduced refractive index changes at electrode surfaces and the chargeconsumed in the process, for example electric deposit of conductivepolymer, its stoichiometry, and the efficiency of the usage of charge inits deposition). It can also selective oxidize and reduce the analyte orligand immobilized. Even though electrochemistry and SPR have been usedtogether in the literature, the current SPR biosensor device has theadditional advantage and convenience to be integrated comparing to thecommercial instruments since in the portable SPR biosensor, all thecomponents are easy to change and to access. Commercial instruments areoften a black box and very hard to change anything.

A further embodiment of the present invention is illustrated in FIG. 20,FIG. 21 and FIG. 22 that incorporates a quartz crystal microbalance(QCM) in a dual SPR/QCM sample holder 610. U.S. Patent Publication No.2005/0003560 (application Ser. No. 10/861,617) to Zeng et al., which ishereby incorporated herein by reference in its entirety, is one exampleof a piezoimmunosensor as the QCM that can be incorporated into the dualSPR/QCM sample holder 610 of the present invention. In this embodiment,the SPR biosensor device 10, 110 is as described previously, except thatthe sample holder 70 is replaced with a dual SPR/QCM sample holder 610.The transparent cover 50 is removably mounted to the prism. The thinfilm of immersion oil (not shown) can be used for optical match betweenthe prism 30 and the transparent cover 50. The thin metallic film 60 isaffixed to the transparent cover 50. The dual SPR/QCM sample holder 610has an o-ring 616 at a first side 610A of the dual SPR/QCM sample holder610 that can be removably sealed against an exposed surface of the thinmetallic film 60. The dual SPR/QCM sample holder 610 holds the o-ring616 firmly against the thin metallic film 60 by the screw 28 threadedthrough the hole 27A (as shown in FIG. 3) in the back plate 27. Whentightened, the screw 28 applies pressure to a spacer plate 611. A usercan secure the dual SPR/QCM sample holder 610 by gripping the knob 29and twisting the screw 28. When the screw 28 is tightened, the dualSPR/QCM sample holder 610, o-ring 616 and thin metallic film 60 form asample chamber 618 (see FIG. 22). A fluid sample can be placed into thesample chamber 618 through an opening 612.

When the screw 28 is tightened against the spacer plate 611, the spacerplate 611 is held away from the dual SPR/QCM sample holder 610 by twonuts 620 protruding from a back side 610B of the dual SPR/QCM sampleholder 610 that are threaded into two screws 622 passing through thedual SPR/QCM sample holder 610. The head 622A of each of the two screws622 are countersunk in a cavity 623 in the front side 610A of the dualSPR/QCM sample holder 610, so that the head 622A of the two screws 622do not interfere with the seal of the o-ring 616 against the thinmetallic film 60. A quartz crystal 630 at the back side 610B of the dualSPR/QCM sample holder 610 provides a back wall of the sample chamber618. The two nuts 620 keep the spacer plate 611 from contacting thequartz crystal 630 at the back side 610B of the dual SPR/QCM sampleholder 610.

The electrode 630 and the electrode 640 are attached to opposite sidesof the quartz crystal 630. These two electrodes are for electric signalsto make the quartz mechanically vibrate at 10 MHz. A reference electrodewire 631 through a reference electrode 632 and out of a top side 610C ofthe dual SPR/QCM sample holder 610. A counter electrode wire 614 passesfrom the sample chamber 618, through a reference electrode hole 642 andout of a top side 610C of the dual SPR/QCM sample holder 610. Thereference electrode, counter electrode and working electrode can be usedfor integration of SPR and/or QCM with electrochemical experiments.

This dual SPR/QCM sample holder 610 design allows multi-techniquesformat (Electrochemistry, SPR and QCM) to be integrated to obtainadditional information to understand and determine the binding eventsbetween the analyte in the solution and the surface receptorsimmobilized on the SPR and/or QCM gold surfaces. It also allows thegeneration of analyte of interest by electrochemistry and to determinewhether the analyte of interest is present in the fluid sample byelectrochemistry Quartz crystal Microbalance or electrochemistry SurfacePlasmon Resonance technique.

EXAMPLES

The portable SPR biosensor device (10, 110) of described in FIG. 1-FIG.3 was used to detect E. coli K12 with a bound lipid mannose-SH samplelayer 66 on the gold metallic film 60. FIG. 15 illustrates the molecularformula of the mannose-SH as a capture reagent bound to gold (Au) thinmetallic film 60 of the surface plasmon resonance biosensor device 10described above. The mannose-SH molecule has a formula of C₂₅H₅₀O₁₀S anda molecular weight (Mr.) of 542.31. A three-dimensional model of themannose-SH is illustrated in FIG. 16. FIGS. 17A and B illustratemannose-SH binding to the gold (Au) surface as the thin metallic film 60of the surface plasmon resonance biosensor device 10, in ethanol. Asillustrated in FIGS. 18A and B, the lectin concanavalin A (ConA) bindsto the mannose-SH bound to the gold (Au) surface. E. Coli K12 can bindto the ConA surface if there is ConA in the liquid, as illustrated inFIGS. 19A and B.

While the present invention is described herein with reference toillustrated embodiments, it should be understood that the invention isnot limited hereto. Those having ordinary skill in the art and access tothe teachings herein will recognize additional modifications andembodiments within the scope thereof. Therefore, the present inventionis limited only by the Claims attached herein.

1.-11. (canceled)
 12. A surface plasmon resonance biosensor system foranalysis of a fluid sample comprising: (a) a support means; (b) acapacitive angle sensor mounted to the support means having a shaft; (c)a rotatable adapter means rotatably mounted on the shaft; (d) a prism ora half cylinder mounted on the rotatable adapter means; (e) a lightsource mounted to the support means, positioned to project a beam oflight through a first side of the prism or half cylinder to a secondside of the prism or half cylinder; (f) a thin metallic film with aninner surface affixed on or adjacent to the prism or half cylinder; (g)a sample holder having a sealing means removably sealed against anexposed surface of the thin metallic film, the sample holder, sealingmeans and thin metallic film defining a sample chamber; (h) a lightresponse element as a transducer to generate an intensity output mountedon a third side of the prism or half cylinder, wherein when the sampleis provided to the sample chamber and the light source projects the beamof light, the capacitive angle sensor measures an incident angle of thebeam of tight upon the metallic film to provide an incident angle outputand the light response element measures light intensity reflected fromthe inner surface of the thin metallic film to provide the intensityoutput; (i) an analog to digital (A/D) data acquisition deviceelectrically connected to the capacitive angle sensor and the lightresponse element for generating a data signal when the sample isprovided to the sample chamber from an incident angle output of thecapacitive angle sensor and an intensity output from the light responseelement; and (j) a personal computer electrically connected to the dataacquisition device to receive and process the data signal generated bythe data acquisition device and provide a surface plasmon resonancespectrum when the fluid sample is provided to the sample chamber. 13.The surface plasmon resonance biosensor system of claim 12, wherein themetallic film is gold.
 14. The surface plasmon resonance biosensorsystem of claim 12, wherein (i) the metallic film is gold and (ii) themetallic film comprises a capture reagent bound to a surface of themetallic film that is internal to the sample chamber.
 15. The surfaceplasmon resonance biosensor system of claim 12, wherein the prism orhalf cylinder is made of glass or plastic.
 16. The surface plasmonresonance biosensor system of claim 12, wherein the light responseelement is a solar cell.
 17. The surface plasmon resonance biosensorsystem of claim 12, wherein the light source is a laser apparatus. 18.The surface plasmon resonance biosensor system of claim 12, wherein therotatable adapter means is a rotatable plate.
 19. The surface plasmonresonance biosensor system of claim 12, wherein the sealing means is ano-ring.
 20. The surface plasmon resonance biosensor system of claim 12,wherein the support means is a mounting table.
 21. The surface plasmonresonance biosensor system of claim 12, further comprising a low speedmotor rotatably engaged with the rotatable adapter means to turn therotatable adapter means during analysis of the sample.
 22. The surfaceplasmon resonance biosensor system of claim 12, wherein the sampleholder is a dual SPR/QCM sample holder which allows simultaneousdetection by both surface plasmon resonance and also quartz crystalmicrobalance (QCM) techniques. 23.-24. (canceled)
 25. The surfaceplasmon resonance biosensor system of claim 12, comprising the prismmounted on the rotatable adapter means.
 26. The surface plasmonresonance biosensor system of claim 12, wherein (i) the capacitive anglesensor measures an outside angle between the beam of light and the prismor half cylinder and (ii) the outside angle is correlated to theincident angle of the beam of light upon the metallic film to providethe incident angle output.
 27. The surface plasmon resonance biosensorsystem of claim 22, wherein the dual SPR/QCM sample holder comprises (i)a first QCM electrode on a quartz crystal surface of the sample holderthat is internal to the sample chamber and that opposes the thinmetallic film, and (ii) a second QCM electrode on a quartz crystalsurface of the sample holder that is external to the sample chamber andthat opposes the first QCM electrode.
 28. The surface plasmon resonancebiosensor system of claim 12, wherein: (i) the thin metallic film servesas a working electrode; (ii) the sample chamber optionally comprises acounter electrode, a reference electrode, or both therein; and (iii) thesample chamber comprising the electrode or electrodes allows performanceof electrochemistry techniques in the sample chamber.
 29. The surfaceplasmon resonance biosensor system of claim 22, wherein: (i) the thinmetallic film serves as a working electrode; (ii) the sample chambercomprises a counter electrode and a reference electrode therein; and(iii) the sample chamber comprising the electrodes allows performance ofelectrochemistry techniques in the sample chamber.
 30. A surface plasmonresonance biosensor system for analysis of a fluid sample, the biosensorsystem comprising: (a) a support table; (b) a capacitive angle sensormounted to the support table, the capacitive angle sensor having ashaft; (c) a rotatable plate rotatably mounted on the shaft; (d) a prismmounted on the rotatable plate; (e) a light source mounted to thesupport table, positioned to project a beam of light through a firstside of the prism to a second side of the prism; (f) a thin goldmetallic film with an inner surface affixed on or adjacent to the prism;(g) a sample holder having a sealing means removably sealed against anexposed surface of the thin gold metallic film, wherein the sampleholder, the sealing means and the thin gold metallic film define asample chamber; (h) a light response element as a transducer to generatean intensity output mounted on a third side of the prism, wherein when asample is provided to the sample chamber and the light source projectsthe beam of light, the capacitive angle sensor measures an incidentangle of the beam of light upon the gold metallic film to provide anincident angle output and the light response element measures lightintensity reflected from the inner surface of the thin gold metallicfilm to provide the intensity output; (i) an analog to digital (A/D)data acquisition device electrically connected to the capacitive anglesensor and the light response element for generating a data signal whenthe sample is provided to the sample chamber from an incident angleoutput of the capacitive angle sensor and an intensity output from thelight response element; and (j) a personal computer electricallyconnected to the data acquisition device to receive and process the datasignal generated by the data acquisition device and provide a surfaceplasmon resonance spectrum when the fluid sample is provided to thesample chamber.
 31. The surface plasmon resonance biosensor system ofclaim 30, wherein (i) the capacitive angle sensor measures an outsideangle between the beam of light and the prism and (II) the outside angleis correlated to the incident angle of the beam of light upon themetallic film to provide the incident angle output.
 32. The surfaceplasmon resonance biosensor system of claim 31, wherein the sampleholder is a dual SPR/QCM sample holder which allows simultaneousdetection by both surface plasmon resonance and also quartz crystalmicrobalance (QCM) techniques, the dual SPR/QCM sample holdercomprising: (i) a first QCM electrode on a quartz crystal surface of thesample holder that is internal to the sample chamber and that opposesthe thin gold metallic film, and (ii) a second QCM electrode on a quartzcrystal surface of the sample holder that is external to the samplechamber and that opposes the first QCM electrode.
 33. The surfaceplasmon resonance biosensor system of claim 30, wherein: (i) the thingold metallic film serves as a working electrode; (ii) the samplechamber optionally comprises a counter electrode, a reference electrode,or both therein; and (iii) the sample chamber comprising the electrodeor electrodes allows performance of electrochemistry techniques in thesample chamber.